EFFECT OF CRYOPROTECTANTS ON ACTIVITY OF SELECTED ENZYMES IN FISH EMBRYOS

Cryoprotectants, which are essential for minimizing cryoinjury during freezing, can be toxic to biological systems. Monohydric alcohols, dim ethyl sulfoxide (Me(2)SO), and ethylene glycol (EG), are known to dena ture enzymes at room temperature. In this study, rosy barb (Puntius co nchonius) and zebra fish (Brachydanio rerio) embryos at cleavage, epib oly, and closure of blastopore stages were exposed to Me(2)SO and EG a t 1.0, 2.0, 3.0, and 4.0 M for 0.25, 0.5, 1.0, 2.0, and 3.0 h at room temperature. The cryoprotectants were then removed and the activities of two glycolytic enzymes, lactate dehydrogenase (LDH) and glucose-6-p hosphate dehydrogenase (G-6-PDH), were determined. Cryoprotectant conc entration and equilibration period had a significant (P < 0.05) effect on the total activity of the two enzymes. In both species the decline in enzymatic activity was more pronounced for G-6-PDH, and EG was mor e toxic than Me(2)SO. Zebra fish embryos were more resistant to cryopr otectant enzymatic denaturation than rosy barb embryos. For both speci es the total LDH activity measured after 3.0 h equilibration in 4.0 M Me(2)SO and EG declined sharply. In zebra fish cleavage, epiboly, and closure of blastopore embryos, the total LDH activity (of control valu e) when Me(2)SO and EG were used was reduced by 70.0 and 86.1, 79.2 an d 83.0, and 57 and 75% for the three embryonic stages, respectively. U nder similar conditions G-6-PDH activity was reduced by 100 and 100, 8 8.3 and 100, and 69.0 and 100%, respectively, for the same embryonic s tages. Similarly for rosy barb embryos, the total LDH activity was red uced by 89.2 and 98.8, 85.0 and 87.1, and 80.6 and 95.4% when equilibr ated in Me(2)SO and EG. In embryos at the closure of blastopore stage, G-6-PDH activity was reduced by 71.2% when equilibrated in 4 M Me(2)S O. The reduction in the total activity of these enzymes was probably d ue to the damage to the perivitelline membrane and blastoderm caused b y the osmotic stress and partial denaturation of the leached enzymes w ithin the perivitelline space. (C) 1995 Academic Press, Inc.